Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 December 2002 - 22 April 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted accoring to OECD standards.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
see "principles of method if other than guideline"
Principles of method if other than guideline:
Deviations:
Animals were tailmarked prior to allocation
Size of cages were 42.5x26.6x15cm
Weekly food intake measured on day 13 instead of day 14
Blood for haematology was taken from 2 animals instead of 5
Determinations that could not be conducted for individual animals are presented in study appendices

None of the deviations were considered to have influenced the validity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetraoctyltin
EC Number:
222-733-7
EC Name:
Tetraoctyltin
Cas Number:
3590-84-9
Molecular formula:
C32H68Sn
IUPAC Name:
tetraoctylstannane
Details on test material:
- Name of test material (as cited in study report): Tetraoctylstannane (TTOT)
- Substance type: Monoconstituent
- Physical state: Colourless liquid
- Analytical purity: 90.79%
- Impurities (identity and concentrations):
Trioctyltin chloride: 8.44%
Dioctyltin dichloride: 0.74%
Monooctyltin trichloride: 0.03%
- Composition of test material, percentage of components: See above
- Purity test date: 11-31 December 2002
- Lot/batch No.: 346272
- Expiration date of the lot/batch: 31 July 2004
- Stability under test conditions: Stable
- Storage condition of test material: At less than -18°C, in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks old
- Weight at study initiation:
Dose range finding-
Males: 276 to 294 grams
Females: 182 to 192 grams
Main study-
Males: 262 to 268 grams
Females: 179 to 186 grams
- Housing:
Premating period: groups of 4/sex were kept in macrolon type 3 cages (42.5 x 26.6 x 15.0 cm)
Mating period: mating pairs were each placed in macrolon type 3 cages
Post-mating period: mated females place singly in macrolon type 3 cages
- Diet (e.g. ad libitum): Commercial rodent diet (Rat & Mouse No. 3, Breeding Diet RM3) from Special Diets Services, Witham, England. Diet was provided ad libitum as a powder in perforated stainless steel cans. These were refreshed once per week.
- Water (e.g. ad libitum): Tap water suitable for human consumption was provided ad libitum in polypropylene bottles
- Acclimation period: 14 days for dose range finding; 7 days for main study


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs light/ 12 hrs dark


Administration / exposure

Route of administration:
oral: feed
Vehicle:
not specified
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Experimental diets were prepared once shortly before the start of the studies; they were stored in a freezer (less than -18°C) until use. The test substance was weighed in a tray of steel and thereafter the tray was rinsed with RM3 food. The weighed test substance was mixed with approximately 3kg weiged RM3 food in the Stephan cutter for 2 minutes. Thereafter, approximately 3 kg of weighed food was used to rinse the Stephan cutter. Mixing was continued in the Lodige for 2 minutes. See Tables 1 and 2 for diet compostions for range-finding and main studies.

DIET PREPARATION
- Rate of preparation of diet (frequency): Once
- Mixing appropriate amounts with (Type of food): RM3
- Storage temperature of food: Less than -18°C in dark

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
From each diet sample, 2.0 g was transferred into a 50 mL Greiner tube. An aliquot of the internal standard solution (monoheptyltin trichloride, diheptyltin dichloride, tripropyltin chloride, and tetrapropyltin in methanol) was added. Subsequently, methanol, acetate buffer solution (pH 4.5), 20% aqueous sodiumtetraethylborate (NaBEt4) solution and hexane (with naphthalene as internal standard) were added to each sample and this mixture was shaken and heated to 60°C. During this step, the organotin chlorides were extracted into the hexane layer. Prior to GC-MS analysis, the hexane layer was washed with 2 mol/L HCl in order to remove (most of) the ethylboron compounds that interfere with the GC-MS analysis. The concentration of each test substance in feed was determined by GC-MS analysis of the hexane extracts. Further details about the derivatization, extraction and analysis are below:

Column: fused silica HP5 MS, 30 m, 0.25 mm ID, 0.25 µm film
Precolumn: fused silica HP5 Ms, 2.5 m, 0.25 mm, 0.25 µm film
Column temperature: after 3 min at 45°C at a rate of 5°C/min to 80°C; then at a rate of 15°C/min to 260°C; 15 min at 260°C
Carrier: helium; 1.5 mL/min constant flow
Injection volume: 1 µL
Injection temperature: start at 60°C, then at a rate of 14.5°C/s to 300°C; 5 min at 300°C
Injection method: splitless
Ionisation: electron impact, 70 eV
Mass range: 60-600 amu
Mass fragments used:
TTOT, m/z= 459; 347; 235
TTPT, m/z= 249

Results & Conclusions:

The test substance TTOT was considered to be homogenously distributed in all diets.
The test substance TTOT was considered to be stable in the diets upon storage at room temperature for 7 days and upon storage at less than -18°C for 55 days.
The content of the test substance TTOT was considered to be close to intended for all diets. Only the 500 mg/kg dose level of the dose-range finding study did not meet the criteria (relative difference from intended concentration was -24%), but when the low recovery of 76% obtained during the validation of TTOT at this dose level is taken into account, the acheived concentration seems to be close to the intended concentration.
In general, the analytical results of TTOT in study samples seem to be influenced by varying efficiencies of TTOT from the diet, resulting from sticking of TTOT to the diet. See Table 3.


Duration of treatment / exposure:
Dose-range finding study: 3 January 2003
Main study:
Males: 28 days- 28 February 2003 to 28 March 2003
Females: 38 to 48 days -28 February 2003 to 7-17 April 2003
Frequency of treatment:
ad libitum in diet.
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1,500 & 7,500 mg/kg in diet.
Basis:
nominal in diet
No. of animals per sex per dose:
Dose-range finding study: Four males and four females per treatment level (five total including control)
Main study: 12 males and 12 females per treatment level (four total inluding control)
Control animals:
yes
Details on study design:
- Dose selection rationale: Based on results of preliminary dose-range finding study
- Rationale for animal assignment (if not random): Randomized computer allocation (proportionally to body weight)
Positive control:
No data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily (morning hours) for both dose-range finding and main studies
- Cage side observations checked in table were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were checked daily for moribundity, abnormalities, reaction to treatment, ill health


BODY WEIGHT: Yes
- Time schedule for examinations:
Dose-range finding study: on day -2 ( for randomization purposes), day 0, 3, 7, 10 and 14 (day of sacrifice)
Main study:
Pre-mating period: on day -2 ( for randomization purposes), day 0, 7, and 13
Mating period: males weighed weekly until sacrifice; females weighed on day 0, 7, 13
Post-mating period: females weighed on day 0, 7, 14, and 21 (during presumed gestation), and on day 1 and 4 of lactation


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: N/A


OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the pre-mating period
- Anaesthetic used for blood collection: Yes, CO2/O2 anaesthesia
- Animals fasted: Yes
- How many animals: 5 rats/sex/group
- Parameters checked: haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell count, prothrombin time, thrombocyte count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of pre-mating period
- Animals fasted: Yes
- How many animals: 5 rats/sex/group
- Parameters checked: fasting glucose, alkaline phosphatase activity, aspartate aminotransferase activity, alanine aminotransferase activity, gamma glutamyl transferase activity, total protein, albumin, ratio albumin to globulin, urea, creatine, total bilirubin, total cholesterol, triglycerides, phospholipids, calcium, sodium, potassium, chloride, inorganic phosphate


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to test initiation; weekly during exposure
- Dose groups that were examined: All
- Battery of functions tested: Functional Observation Battery (FOB)


OTHER: Parturition and litter evaluation; litter size, sexes and pup weight; pathology of pups; fetility and reproductive performance
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, ovaries, uterus, testes, epididymides, seminal vesicles, prostrate, other organs and tissues
HISTOPATHOLOGY: Yes, adrenal tissue, lymph nodes (axillary and mesenteric), bone marrow, brain, caecum, colon, duodenum, eyes, heart, ileum, jejunum, lungs, kidneys, liver, parathyroids, Peyer's patches, pituitary, rectum, sciatic nerve, spinal cord, spleen, stomach, thymus, thyroids, trachea, urinary bladder
Other examinations:
None
Statistics:
Clinical findings were evaluated by Fisher's exact probability test
Body/organ weight, food consumption were evaluated by one-way ANOVA followed by Dunnett's multiple comparison test
Mated/pregnant females evaluated with Fisher's exact probability test
Implantation sites, live and dead pups were evaluated by Kruskal-Wallis nonparametric ANOVA with Mann-Whitney U-test.
Blood counts, chemistry values, organ weights evaluated by one-way ANOVA followed by Dunnett's multiple comparison tests
Reticulocytes and relative differential white blood cell counts evaluated by Kruskal-Wallis non-parametic ANOVA followed by Mann-Whitney U-tests.
Continuous FOB parameters assessed via Kruskal-Wallis ANOVA; categorical FOB parameters were evaluated by Pearson chi-square analysis

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY:
No mortalities were observed.
No clinical signs were observed in the males from the start of the study until sacrifice (Table 7).
During the premating period, female C569 of the mid-dose group was sparsely haired and in female D585 of the high-dose group a kinked tail was observed. Animal D585 showed a kinked tail until sacrifice. Exopthalmus was observed after orbital puncture in female C565 of the mid-dose group and female D575 of the high-dose group. This observation lasted until GD 0 for animal D575 and GD 3 for animal C565. Thereafter, panophthalmitis was observed in both animals until sacrifice. During the gestation period, animals A509 (control group, GD 20), C553 and C569 (mid-dose group, GD 1 and GD 20, respectively) were sparsely haired. During the lactation period, the following animals were sparsely - haired: animal A509 (control, PN 0-5) and C551 and 553 (mid-dose, PN 0-4).


BODY WEIGHT AND WEIGHT GAIN:
Mean body weight of the male animals of the tetraoctylstannane-treated groups was comparable to the control group. Body weight change of the male animals was statistically significantly decreased in the high-dose group from days 0-7 and 7-13 and in the low- and mid-dose groups from day 7-13. The effect on body weight change in the tetraoctylstannane-treated groups was not dose-related and in addition compensated during the other periods. Furthermore, no effect on mean body weight was observed. For that reason this finding was considered of no toxicological significance.
During the premating, gestation and lactation periods, the mean body weight and body weight change of the female animals of the tetraoctylstannane-treated groups was comparable to the control group.


FOOD CONSUMPTION AND COMPOUND INTAKE:
During the premating period, mean food consumption expressed as g/animal/day of the female animales of the low-, mid-, and high-dose group was statistically significantly decreased from days 0-7 and expressed as g/kg body weight/day in the mid- and high-dose group from days 0-7.

FOOD EFFICIENCY: N/A

WATER CONSUMPTION AND COMPOUND INTAKE: N/A

OPHTHALMOSCOPIC EXAMINATION: N/A

HAEMATOLOGY:
The only statistical difference observed between the tetraoctylstannane-treated groups and the control group was the decrease in mean corpuscular haemoglobin concentration in the female animals of the low-dose group. In the absence of similar findings in the mid- and high-dose groups this finding is considered an incidental finding.


CLINICAL CHEMISTRY:
The only statistical difference observed between the tetraoctylstannane-treated groups and the control group was increase in albumin/globulin ratio in females of the high-dose group. Since there was no significant difference in total protein or albumin concentration, the change in the calculated albumin/globulin ratio was considered of no toxicological relevance.

URINALYSIS: N/A

NEUROBEHAVIOUR:
Treatment-related changes were not observed during the neurobehavioural testing of males and females at arena testing during the study and FOB and motor activity assessment at the end of the study. Therefore, no evidence was obtained for a neurotoxic potential of the test substance.

ORGAN WEIGHTS: In the high-dose group, the absolute thymus weight of the male animals was statistically significantly decreased. The absolute (0.049 high-dose versus 0.121 g control) and relative (0.236 high-dose versus 0.599 g/kg body weight control) thymus weight of the female animals of the high-dose group was statistically significantly decreased.

GROSS PATHOLOGY:
Male animals were sacrificed on day 33. In high-dose group, the absolute thymus weight of the male animals was statistically significantly decreased. Relative thymus weight of the male animals of this group was lower (not statistically significantly) than the control group (0.785 high-dose versus 1.154 g/kg body weight control). Female animals with a litter were sacrificed on PN 4 or 5. The absolute (0.049 high-dose versus 0.121 g control) and relative (0.236 high-dose versus 0.599 g/kg body weight control) thymus weight of the female animals of the high-dose group was statistically significantly decreased.
No other effects on organ weights (absolute and relative) were observed.

Male animals were sacrificed on day 33. Female animals with a litter were sacrificed on PN 4 or 5; non-pregnant animals were sacrificed on GD 24. At necropsy an increased incidence of accumulation of fluid (hydrometra), was seen in the uteri of three non-pregnant low-dose females. Two of these animals showed swelling of the cervix as well. Since hydrometra of the uterus is part of common background pathology and a dose-response relationship was absent, no significance was attached to this finding. The other macroscopic observations are common findings in rats of this strain and age and occurred only incidentally.
The decreased size of the thymus was not reported as a gross lesion as decreases in thymus weights were considered to reflect the diminished size of the thymus in individual animals more accurately.


HISTOPATHOLOGY (NON-NEOPLASTIC):
Examination of the thymus revealed very severe lymphoid depletion in 5/5 high-dose females and moderate lymphoid depletion in 1/5 high-dose males. Lymphoid depletion was characterized by a decrease in the size of thymic lobules because of an extensive loss of cortical and medullary small lymphocytes.


HISTOPATHOLOGY (NEOPLASTIC):
In the mesenteric lymph nodes of 3/5 high-dose females and 1/5 mid-dose female, the presence of clusters of swollen yellowish macrophages in the medullary cords, and occasionally in the paracortical areas, was conspicuous (denoted as very slight to slight macrophage accumulations). The medullary cords had been enlarged by these macrophage accumulations.

Effect levels

Dose descriptor:
NOAEL
Effect level:
ca. 1 500 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: General toxicity

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Mean organ weights relative to terminal body weight (g/kg body weight)- female

 

Control

500mg/kg

1500 mg/kg

7500 mg/kg

Terminal body weight (g)                  

209.1

207.6

217.4

208.5

Adrenals

0.308

0.282

0.282

0.273

Brain

8.493

8.143

8.246

8.020

Heart

3.465

3.465

3.282

3.354

Kidneys

6.446

5.842*

5.916*

6.049

Liver

35.716

38.563

39.991

36.467

Spleen

2.399

2.051

1.947

2.029

Thymus

0.599

0.534

0.498

0.236#

* = p < 0.05

# = p < 0.001

Table 2: Mean organ weights absolute (g)- males

 

Control

500 mg/kg

1500 mg/kg

7500 mg/kg

Terminal body weight

356.0

350.6

352.3

342.9

Testes

3.27

3.252

3.244

3.235

Epididymides

1.213

1.186

1.187

1.178

Adrenals

0.049

0.050

0.047

0.045

Brain

1.871

1.870

1.815

1.820

Heart

1.089

1.115

1.079

1.097

Kidneys

1.988

2.011

2.101

2.009

Liver

12.944

12.147

12.492

12.325

Spleen

0.638

0.672

0.676

0.644

Thymus

0.411

0.377

0.402

0.269*

* = p < 0.05

Applicant's summary and conclusion

Conclusions:
Based on the observed effects in the high-dose animals, decrease in thymus weight (male and female animals) and microscopic findings in the thymus (female animals), the No Observed Adverse Effect Level (NOAEL) for general toxicity is established at the mid-dose level (1,500 mg/kg diet which is equivalent to 86-99 mg/kg body weight/day for the male animals and to 80-141 mg/kg body weight/day for the female animals).
As no significant reproductive effects were observed in the tetraoctylstannane-treated groups, the NOAEL for reproductive toxicity was established at the high-dose level (7,500 mg/kg diet which is equivalent to 445-480 mg/kg body weight in the male animals and 426-624 mg/kg body weight in the female animals).
Executive summary:

The substance was tested in an experiment conducted in compliance with GLP and in accordange with OECD guideline 422. The objective of this study was to provide data on the possible reproductive and developmental effects of tetraoctylstannane [CAS # 3590-84-9] after oral administration via the diet to Wistar rats of both sexes. The study was combined with a repeated dose toxicity study, rats were fed diets containing 0, 100, 500, 2000 and 10,000 mg/tetraoctylstannane/kg diet for 14 days. In the main study, rats were fed diets containing 0, 500, 1500, 7500 mg tetraoctylstannane/ kg diet for up to 33 days (males) or during 2 weeks premating, mating, gestation and up to day 4 or 5 of lactation (females).

Dose-range finding study:

Administration of tetraoctylstannane via the diet up to a level of 2000 mg/kg diet for a period of 14 days was well tolerated by the rats. The only treatment-related change was an effect on thymus weight in the females and males of the 10,000 mg/kg group.

Main study:

No treatment-related mortalities or clinical signs were observed. In male animals tested after 4 weeks of treatment and in female animals tested on postnatal day (PN) 4, no changes indicative of neurotoxic potential of the test substance were observed in the neurobehavioural observation and motor activity assessment.

Administration of tetraoctylstannane via the diet (during the first week of the premating period) resulted in a decrease in food consumption in the female animals of all tetraoctylstannane-treated group when compared to the control. During the study no other differences in food consumption were observed in male or female animals of the tetraoctylstannane-treated groups.

At necropsy, three of the non-pregnant females of this group showed hydrometra of the uterus, which was confirmed upon microscopic examination by luminal dilation. In the absence of similar findings in the mid- and high-dose groups this finding is considered to be incidental.

No statistically significant effects were observed on the female fecundity, male and female fertility, gestation index, pre-coital time, number of females with liveborn pups, sex ratio, number of females with stillborn pups, number of females with only stillborn pups, post-implantation loss, number of pups delivered, number of liveborn pups, number of live pups/litter, number of stillborn pups, pup mortality PN 4, pup weight PN 1 and 4 and number of runts.

In the high-dose group, the absolute thymus weight of the male animals was statisically significantly decreased. Relative thymus weight of the male animals of this group was decreased (approx. 32%), but not statistically significantly. In the females of the high-dose group, the absolute and relative thymus weights were statistically signficantly decreased by appox. 60%. No other effects on organ weights (absolute or relative) were observed.

In conclusion, based on the observed effects in the high-dose animals, decrease in thymus weight (male and female animals) and microscopic findings in the thymus (female animals), the No Observed Adverse Effect Level (NOAEL) for general toxicity is established at the mid-dose level (1500 mg/kg diet which is equivalent to 86-99 mg/kg body weight/day for the male animals and to 80-141 mg/kg body weight/day for the female animals.

In addition, as no significant reproductive effects were observed in the tetraoctylstannane-treated groups, the NOAEL for reproductive toxicity was established at the high-dose level (7500 mg/kg diet which is equivalent to 445-480 mg/kg body weight in the male animals and 426-624 mg/kg body weight in the female animals.